Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21759


Recent Submissions

Now showing 1 - 17 of 17
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    (2014-03) Silverman, Sean; Kim, Byung-Jin; Wordinger, Robert J.; Clark, Abbot F.
    We are using a mouse model whereby blood flow to the eye is blocked by raising the pressure in the eye in order to mimic damage caused by glaucoma. Our interest is to see how levels of C1q, a protein typically associated with the immune system as well as injury responsive cells of the eye are changed. Purpose (a): The complement cascade has become of increasing interest in several neurodegenerative diseases, including glaucoma, a leading cause of blindness. C1q has been observed as one of the earliest upregulated genes in the optic nerve head, the initial site of glaucoma injury preceding pathological changes. Here we use a glaucoma-like model of retinal ischemia/reperfusion (I/R) to mimic clinical changes in visual function and cellular loss. Methods (b): Deeply anesthetized C57BL/6J received a cannula to the anterior chamber of their left eye, through which their intraocular pressure (IOP) was raised to 120mmHg for 60 minutes leading to complete retinal ischemia. The cannula was then removed and blood flow was naturally reperfused. The right eye was uninjured as a contralateral control. Mice were sacrificed and enucleated at 3, 7, 14, 21, and 28 days. Eyes were fixed in 4% PFA and frozen for immunofluorescence or in situ hybridization studies. Microglia and astrocytes were identified using Iba1 and GFAP, respectively. Quantifications were performed using ImageJ Analysis software(NIH). Results (c): Initial changes in C1q expression were observed as early as 72 hours following injury, with a nearly two-fold increase compared to uninjured controls. Upregulated C1q was observed only in the ganglion cell (GCL) and inner plexiform (IPL) layers. Maximum intensity of C1q expression was observed 14 days post injury. Fluorescent in situ hybridization (FISH) studies reveal primarily microglia, not astrocytes, colocalized with expression of C1q in the retina. Conclusions (d): Following retinal I/R injury, C1q expression is actively upregulated, which appears to spatio-temporally correlate with changes in microglial, astrocyte, and Mueller cell homeostasis. Our FISH studies identify microglial cells as the primary producers of C1q following I/R injury. This suggests the elevated levels of C1q may stimulate astrocyte activation. There appears to be an interplay between microglia and astrocytes, both of which have been directly implicated in neurodegenerative diseases, including loss of RGCs in glaucoma. We propose C1q is an integral part of this mechanism, and by removing C1q we hope to preserve visual function and prevent degeneration in the visual system following injury.
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    (2014-03) McGrady, Nolan R.; Minton, Alena Z.; Krishnamoorthy, Raghu R.
    Glaucoma is the leading cause of blindness in developed countries and is the second leading cause of blindness worldwide. The most common and currently only treatable symptom associated with glaucoma is an increase in intraocular pressure (pressure inside the eye). Rodent models have been routinely used to understand the effects elevated pressure has on the eye, and this study focuses on the changes in expression of a protein molecule (endothelin A receptor) within the retina due to elevated intraocular pressure in a rat model of elevated intraocular pressure. Purpose (a): The endothelin system of peptides and their receptors have been implicated for their neurodegenerative role in glaucoma. The purpose of this study was to determine changes in ETA receptor expression within the retina in the Morrison’s elevated IOP model of glaucoma in rats. Methods (b): IOP was elevated in the left eye of adult male retired breeder Brown Norway rats using the Morrison’s model of glaucoma (by injection of hypertonic saline through episcleral veins) while the contralateral eye served as the control. The rats were maintained for two to four weeks following IOP elevation and sacrificed. Retinal sections were obtained from both control and IOP-elevated eyes, and analyzed for changes in ETA receptor expression using immunohistochemistry. ETA receptor immunostaining was co-localized with β-III-Tubulin, which is selectively expressed in retinal ganglion cells. Results (c): After two weeks, rat eyes with IOP elevation showed an increase in immunostaining for ETA receptors in several retinal layers including the inner and outer plexiform layers with a modest increase in the retinal ganglion cell layer. Following four weeks of IOP elevation, ETA receptor expression was modestly increased in the inner and outer plexiform layers of the retina, compared to that in the corresponding contralateral eyes. Conclusions (d): Elevated intraocular pressure results in a time-dependent change in ETA receptor expression. Increased ETA receptor expression is associated with neurodegenerative changes in glaucoma.
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    (2014-03) Kirkland, Kyle A.; Bermudez, Jaclyn Y.; Mao, Weiming; Clark, Abbot F.
    Purpose: Glaucoma is a leading cause of irreversible blindness in the world. TGF-β2 is elevated in glaucoma eyes and is an important factor in the extracellular matrix (ECM) metabolism of human trabecular meshwork (HTM) cells, leading to increased intraocular pressure (IOP). Both LOX and TGM2 enzymes are important in cross-linking the ECM in HTM cells. As TGF-β2 up regulates LOX and TGM2 expression, the aim of this experiment is to determine and quantify the amount of LOX- and TGM2-induced ECM crosslinking in glaucomatous trabecular meshwork cells (GTM). Methods: LOX and TGM2 cDNAs were obtained and amplified by PCR with specifically designed primers and isolated by gel electrophoresis. The cDNAs were ligated into the pGEM-T plasmid vector and cloned into E. coli gold cells. After sequencing, the genes were restriction digested and ligated to a pacAd5 vector to generate adenovirus expression vectors, which have a high selectivity for TM cells. The pacAd5 vectors will be used to transduce the GTM3 cell line. The plasmid expression vectors will also be transfected into GTM3 cells. Western immunoblot analysis will be utilized to evaluate the LOX and TGM2 protein expression and ECM crosslinking in GTM cells. Results: LOX and TGM2 plasmid vectors were successfully cloned and sequenced and are now being used to transfect GTM cells. LOX and TGM2 adenoviral vectors are being prepared. We expect that increased expression of LOX and TGM2 enzymes through both transduction and transfection will induce a greater amount of crosslinking in the GTM cells. Conclusions: TGF-β2 raises IOP by mechanisms that are still under investigation. One potential mechanism is increased ECM cross-linking via TGF-β2 induction of LOX and TGM2 gene expression. Successful transduction and LOX and TGM2 expression in cultured GTM3 cells will allow us to directly test the roles of LOX and TGM2 on the regulation of IOP using an ex vivo bovine ocular perfusion culture model.
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    (2014-03) Mueller, Brett H.; Park, Yong; Ma, Hai-Ying; Yorio, Thomas
    Purpose (a): Calcium influx through postsynaptic NMDA receptors has been shown to stimulate a number of key pro-survival genes; however, prolonged stimulation has been shown to have excitotoxic effects leading to apoptosis in neurons. Previous studies have shown a rapid dephosphorylation of CREB in primary hippocampal neurons treated for 1-2 h with100µM NMDA . It is hypothesized that the activation of CREB-specific phosphatases is one of the main pathways that cause apoptosis during NMDA excitotoxicity. The current study investigated the role of NMDA stimulation on the phosphorylation of CREB in primary RGCs, and assessed if NMDA overstimulation caused excitotoxic changes similar to those seen in primary hippocampal neurons. In addition, the occurrence of NMDA excitotoxicity in bipolar and photoreceptor cells was also investigated. Methods (b): Purification and culture of RGCs were performed by sequential immunopanning using Thy 1 antibody from P3-P7 Sprague-Dawley rats. Mixed retinal cultures that remained following isolation of RGCs from the retina were plated once the RGCs were separated and purified. Calcium imaging was used to measure the intracellular changes in calcium following treatment of cells with 100µM NMDA. Western blots were performed to determine signaling pathways linked to NMDA induced cell survival or excitotoxicity. Calcein AM and ethidium homodimer were used to quantify cell survival and cell death. Cells were also subjected to a trophic factor deprivation insult for 6 hours and 24 hours. Results (c): Treatment of primary RGCs with NMDA (100 µM) for 6h caused a greater than 2-3 fold induction of the transcription factor pCREB. MK801 (NMDA antagonist) completely abolished endogenous levels of pCREB and blocked NMDA induction of pCREB. NMDA (100 µM) treatment for 6 and 24 hrs under trophic factor deprivation, protected RGCs from trophic factor deprivation induced cellular death. The mixed retinal cultures (retinal cells without RGCs) had an opposite effect, where the levels of pCREB were diminished and the neurons died when treated with 100 µM of NMDA. Conclusions (d): The data suggests that NMDA signaling is essential for RGC survivability and blocking calcium ion influx through this receptor by the NMDA blocker, MK801 can be detrimental to RGC function and survival. These results also demonstrate that primary RGCs behave differently than other neurons in the retina, and are not susceptible to NMDA excitotoxicity.
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    (2014-03) Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan
    Acanthamoeba keratitis (AK) is a vision-threatening disease caused by pathogenic strains of Acanthamoeba. The main risk factors for AK are wearing eye contact lenses, corneal injuries, and contact with contaminated water. Diagnosis of AK is not straightforward and treatment is very demanding because of Acanthamoeba cysts resist to most antimicrobial agent. Our project is based on the observations that the innate immune system plays an important role in AK, and exploring the sequential pathogenic cascades of AK and targeting the therapeutic approaches to determine new remedies to prevent and treat AK. We have shown that Acanthamoeba trophozoites secrete cytopathic serine proteases, MIP-133 and aPA. Role of MIP-133 in the pathogenesis of AK via cPLA2α pathway has been explored; however, aPA interaction to corneal epithelial cells in disease progression is still unknown. In the present study, we have shown that aPA specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway and PAR2-antagonists may be a therapeutic target in AK. Purpose (a): Acanthamoeba plasminogen activator (aPA), is a serine protease elaborated by Acanthamoeba trophozoites, facilitates invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA induces proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptor PAR2 pathway. Methods (b): A. castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system and aPA activity was determined by zymography assays. HCE cells were incubated with or without aPA (100µg/ml), PAR1-agonists (Thrombin, 10µM; TRAP-6, 10µM), and PAR2-agonists (SLIGRL-NH2, 100µM; AC55541, 10µM) for 24 hours. Inhibition of PAR1 and PAR2 involved pre-incubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH79797, 60μM) and PAR2 (FSLLRY-NH2, 100μM) and then incubated with or without aPA, Thrombin, TRAP-6, SLIGRL-NH2, and AC55541 for 24 hours. Expression of PAR1 and PAR2 was examined by qRT-PCR, flow cytometry, and immunocytochemistry. IL-8 expression was quantified by qRT-PCR and by ELISA. Results (c): PAR1 and PAR2 surface protein were expressed in HCE cells. aPA and PAR2-agonists significantly upregulated PAR2 expression (~1-2 times) (P<0.05). PAR2-antagonist significantly inhibited aPA and PAR2-agonists-induced PAR2 expression (~2-5 times) (P<0.5) in HCE cells. PAR1-agonists, but not aPA, significantly upregulated PAR1 expression, which was significantly inhibited by PAR1-antagonist (~30-45 times) in HCE cells. aPA and PAR2-agonists, but not PAR1-agonists, stimulated IL-8 production, which is significantly diminished by PAR2-antagonist (~2-10 times) (P<0.5). PAR1-antagonist did not diminish aPA-induced IL-8 production in HCE cells. Conclusions (d): aPA specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway and PAR2-antagonists may be a therapeutic target in AK.
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    (2014-03) Medina-Ortiz, Wanda E.; Wordinger, Robert J.; Clark, Abbot F.
    Glaucoma is one of the leading causes of blindness, set off generally by changes on eye pressure and tissue modifications. One of the common tissue modifications includes induction of extracellular proteins expression on the human eye trabecular meshwork. This study provides important information on one of the tissue changes caused by a factor involved in glaucoma, Transforming Growth Factor-beta 2, the induction of the expression of the extracellular protein fibronectin. Fibronectin is a protein that can be found in two types of forms, a plasma soluble form and a cellular insoluble form. This study showed how interaction with cellular fibronectin form can affect normal human trabecular meshwork cells. This type of tissue modification may be part of the trabecular meshwork changes that can lead to glaucoma. Understanding the tissue changes and factors affecting the eye conditions leading to glaucoma will help in the discovery of agents protecting the eye from glaucoma damage. Purpose (a): The expression of cellular fibronectin isoforms (cFN) are induced by transforming growth factor-beta 2 (TGF-β2) in cultured human trabecular meshwork (TM) cells, and TGF-β2 expression is elevated in glaucomatous TM tissues. Cellular interaction with cFN isoforms can affect extracellular matrix (ECM) homeostasis, as well as the cellular interaction and response to the surrounding microenvironment. Our purpose is to determine the impact of the interaction of normal HTM (NTM) cells with cFN isoforms on the metalloproteinase (MMP) expression and the TGF-β2 signaling pathway. Methods (b): NTM cell strains were cultured for up to 2 days on surfaces coated with cFN, and the responses were compared to control uncoated surfaces. In addition, to show that the EDA domain of cFN was involved, NTM cells were cultured in the presence of anti-EDA antibodies. Changes on gene and protein expression and cellular distribution of MMPs and TGF-β2 signaling pathway components were analyzed using qRT-PCR, Western immunoblots and immunocytochemistry. Results (c): NTM cell strains exposed to cFN isoforms significantly decreased MMP-1 and MMP-3 expression, and this effect was blocked by anti-EDA pre-incubation. cFN significantly altered the expression of TGF-β2 signaling pathway components, including regulatory and inhibitory SMADs. The phosphorylation and nuclear translocation of regulatory SMADs also was increased, indicating activation of the TGF-β signaling pathway. Conclusions (d): Our results demonstrate that NTM cell interactions with cFN isoforms decreases levels of critical components involved in ECM homeostasis. Furthermore, we show that interaction with cFN affects different TGF-β2 signaling components further activating this signaling pathway. In summary, our data suggest that interaction of NTM cells with a glaucoma-like ECM (i.e. cFN) further exacerbates TGF-β2 signaling leading to decreased ECM turnover and fibrosis.
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    (2014-03) Liu, Yang; Sharma, Tasneem; Wordinger, Robert J.; Gorbatyuk, Marina S.; Clark, Abbot F.
    We delivered GRP78/BiP gene into the mouse eye and promoted retinal ganglion cell survival after optic nerve injury. This study suggests a potential therapeutic target for central nervous system neurodegenerative diseases such as glaucoma and optic nerve trauma. Purpose (a): Optic nerve injury triggers endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR), leading to retinal ganglion cell (RGC) degeneration. Glucose-regulated protein (GRP78/BiP) is a sensor of ER homeostasis and plays a role in ER stress alleviation. In this study, we evaluated the involvement of GRP78/BiP in RGC degeneration induced by optic nerve crush (ONC) and the neuroprotective effects of gene delivery of GRP78/BiP. Methods (b): ONC was performed unilaterally in adult BALB/cJ mice. The expression of GRP78/BiP was evaluated by real time PCR and fluorescent in situ hybridization (FISH). To evaluate the potential neuroprotective effect of BiP , an AAV2 vector harboring the human BiP gene (AAV2-hBiP) or green fluorescent protein (AAV2-GFP) (2 x 109 P) was intravitreally injected 4 weeks prior to the ONC. Seven and fourteen days after the ONC, RGC survival was determined by RBPMS immunofluorescence staining of retinal flat mounts. Retinal function was assessed using full field flash ERG. Expression of UPR related proteins was evaluated by western blotting. Results (c): Three days after ONC, GRP78/BiP expression was significantly up-regulated in RGCs (p<0.01). Intravitreal administration of AAV2-hBiP significantly reduced RGC loss at 7 and 14 days post-ONC compared to AAV2-GFP injected group (n=5, p<0.01). ERG analysis showed partial protection of pSTR amplitudes in AAV2-hBiP injected eyes (n=5, p<0.05). Retina levels of cleaved ATF6 in AAV2-hBiP injected eyes were much lower than those of AAV2-GFP injected eyes. Conclusions (d): Gene delivery of GRP78/BiP promotes RGC survival and preserves RGC function following optic nerve injury. This study suggests a potential therapeutic target for central nervous system neurodegenerative diseases.
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    (2014-03) Minton, Alena Z.; He, Shaoqing; Ma, Hai-Ying; Krishnamoorthy, Raghu R.
    Glaucoma is a group of eye conditions that, if left untreated, can result in blindness. It is commonly associated with an increased pressure inside the eye, known as intraocular pressure or simply IOP. As the pressure builds up inside the eye, it causes damage to the optic nerve, which in turn results in the death of retinal ganglion cells (RGCs). Studies from our lab and others have shown that endothelin 1 (ET-1), the potent vasoactive peptide, contributes to glaucoma. Currently, our lab is interested in understanding the role of the endothelin receptor B (ETB) in glaucoma. We are using rats that do not have ETB receptor (ETB KO rats) and those that have the receptor (WT rats). To mimic glaucoma, the high salt solution is injected into the special vein in the eye (episcleral vein). This causes the build up of pressure inside the eye within 7 to 10 days. This model of glaucoma is called the Morrison’s ocular hypertension model. Previously, we found that IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in ETB KO rats. In addition, pathological changes in the optic nerve were greatly reduced in ETB KO rats, as compared to those in WT rats. To find out the molecular mechanisms responsible for the death of RGCs, we elevated the pressure inside one eye of adult WT and ETB KO rats, while the contralateral eye served as control. After 2 weeks of IOP elevation, retinal sections were obtained and stained with specific antibodies to detect the levels of c-Jun (the member of the activator protein-1 (AP-1) family) and Bax (protein involved in cell death). We found that WT rats have higher levels of c-Jun and Bax in the retina (especially in the ganglion cell layer), as compared to ETB KO rats. Interestingly, using the Promo 3 software, we found 15 binding sites for members of the AP-1 family of proteins on the rat 1.95 kb upstream promoter region of Bax. Therefore, the transcription factor c-Jun may be an upstream regulator of Bax. In conclusion, transcription factor AP-1 could be involved in the elevation of the ETB receptor levels in the Morrison's model of glaucoma. Conversely, deletion of the ETB receptor results in the lower expression of c-Jun. Taken together, there may be a reciprocal relationship between the AP-1 and ETB receptors. Purpose (a): Previously, our lab has demonstrated that increased levels of ETB receptors contribute to the death of retinal ganglion cells (RGCs) and degeneration of optic nerve axons in the Morrison's elevated intraocular pressure (IOP) model of glaucoma in rats. Moreover, these pathological changes were greatly attenuated in ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, an increase in ETB receptor levels in RGCs, following 2 weeks of IOP elevation in Brown Norway rats, was shown to be associated with increased expression of c-Jun, a member of the activator protein-1 (AP-1) family. The current study was aimed at investigating whether the increased expression of c-Jun observed in wild type rats is reduced in ETBreceptor-deficient Wistar Kyoto rats subjected to the Morrison’s model of glaucoma. The status of another apoptotic protein, Bax, was also assessed in these rats. Methods (b): IOP was elevated in one eye of adult wild type and ETB receptor-deficient transgenic Wistar Kyoto rats using the Morrison’s method (injection of hypertonic saline through episcleral veins), while the contralateral eye served as control. After IOP was elevated, rats were maintained for 2 weeks and sacrificed. Retinal sections were obtained and stained with specific antibodies to detect the expression of c-Jun and Bax by immunohistochemistry. In addition, retinal sections were immunostained using an antibody to βIII-tubulin, which is selectively expressed by RGCs in the retina. Images were taken using Zeiss LSM-510 confocal microscope with Z-scan. Results (c): Immunohistochemical analysis showed that IOP elevation for 2 weeks caused increased expression of c-Jun and Bax mainly in the ganglion cell layer (GCL) of wild type transgenic Wistar Kyoto rats as compared to ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, using the Promo 3 software, we found 15 binding sites for members of the AP-1 family of proteins on the rat 1.95 kb upstream promoter region of Bax. Therefore, the transcription factor c-Jun may be an upstream regulator of Bax (pro-apoptotic factor). Conclusions (d): Transcription factor AP-1 could be involved in the elevation of the ETB receptor levels in the Morrison's model of glaucoma. Conversely, deletion of the ETB receptor results in the downregulation of c-Jun. Taken together, there may be a reciprocal feedback loop between the AP-1 and ETB receptors.
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    (2014-03) Bermudez, Jaclyn Y.; Webber, Hannah; Cheng, Yi-Qiang; Clark, Abbot F.; Mao, Weiming
    Glaucoma is a leading cause of blindness in the U.S. and worldwide. The primary risk factor of primary open angle glaucoma (POAG), the major type of glaucoma, is elevated intraocular pressure (IOP). IOP elevation in glaucoma patients is due to glaucomatous insults to the trabecular meshwork (TM) and compromised TM function. Therefore, it is important to study how glaucoma-associated growth factors in the TM are regulated. We investigated how heritable changes in gene activity regulate the TM without altering the DNA sequence. Purpose (a): Glaucoma is a leading cause of blindness in the U.S. and worldwide. This disease leads to progressive, irreversible damage to the optic nerve and visual function. The primary risk factor of primary open angle glaucoma (POAG), the major type of glaucoma, is elevated intraocular pressure (IOP). IOP elevation in glaucoma patients is due to glaucomatous insults to the trabecular meshwork (TM) and compromised TM function, which increase aqueous humor outflow resistance. In the glaucomatous TM (GTM), there is excessive extracellular matrix (ECM) protein deposition. Many studies have suggested that cell signaling pathways, such as the transforming growth factor beta (TGF-β) and Wnt signaling pathways, play key roles in TM homeostasis.The growth factors that are associated with these pathways, including TGFβ2, Gremlin and sFRP1, are found to be at higher levels in the GTM cells compared to normal TM cells. Little is known about the role of epigenetics in regulating glaucoma-associated growth factors in the TM. One of the major epigenetic regulatory mechanisms is histone acetylation.We hypothesize that histone acetylation is responsible for the increased expression of glaucoma associated factors in the TM. Methods (b): Primary human TM cell cultures were treated with 10nM Thailandepsin (TDP-A), a histone deacetylase inhibitor (HDACi), or 1% DMSO as vehicle control for 4 days. Cells were harvested for qPCR to compare gene expression levels or for ChIP assays to compare promoter associated histone acetylation status. We also treated paired perfusion cultured bovine anterior segments with DMSO or TDP-A for 7 to 10 days. The IOP change of the treated bovine eyes was monitored and recorded. Data were analyzed by using Student’s t-test or one-way ANOVA. P values less than 0.05 were considered significant. Results (c): TDP-A significantly elevated the expression of sFRP-1 and TGFβ2 (n=3, p2 as well as elevated IOP. Conclusions (d): Histone acetylation may play an important role in the dysregulation of growth factors in the TM. This mechanism provides a unique opportunity to elucidate the etiology of POAG. Also, TDP-A is a potent HDACi that can be used as a powerful tool in glaucoma research.
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    (2014-03) Park, Yong H.; Mueller, Brett H.; McGrady, Nolan; Ma, Hai-Ying; Dibas, Adnan; Yorio, Thomas
    Glaucoma is an age-related disease that affects nearly 70 million people worldwide. It is characterized by damage to the cells in the back of the eye which eventually die and cause gradual vision loss. The mechanism to how glaucoma occurs is yet unknown but there are many speculations. A protein molecule called the AMPA receptor is speculated to play a role in glaucoma by causing the death of these cells in the back of the eye. In our study, we are isolating the cells from the back of the eye of rats to study the role of the AMPA receptor and how it truly functions. Understanding basic functions of this protein molecule can one day help us develop drugs targeting AMPA receptors and therefore possibly protecting the dying cells in glaucoma. Purpose (a): The ionotropic glutamate receptors (iGluR) have been hypothesized to play a role in glaucoma pathogenesis by mediating excitotoxic death of retinal ganglion cells (RGC). Previous studies on iGluR in the retina have been focused on two broad classes of receptors: NMDA and non-NMDA receptors including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and Kainate receptor. In this study, we examined the specific excitotoxic effects of activation of the AMPAR in RGCs in-vitro. Methods (b): Purified rat RGCs were isolated from P3-P5 Sprague-Dawley rats by a double immunopanning technique using an antibody to Thy 1.1. RGCs were cultured for 7 days before s-AMPA (100μM) treatments. s-AMPA excitotoxicity was determined by Caspase3/7 luciferase activity assay, immunoblot analysis for α-fodrin and Live (calcein AM)/Dead (ethidium homodimer-1) assay. Gap-43 expression was assessed by immunocytochemistry. Results (c): Treatment of cultured RGCs with s-AMPA (100μM) for 24, 48 and 72h, both in the presence and absence of trophic factors (BDNF and CNTF), did not alter caspase 3/7 activity and cleavage of α-fodrin (neuronal apoptosis marker), compared to untreated controls. A significantly higher (p<0.05) cell survival of RGCs (85.3±1.5% alive cells) was observed after a 72h treatment with 100μM s-AMPA compared to control untreated RGCs (74.8±3.1% alive cells). Quantification of s-AMPA (100μM) – mediated excitotoxicity in purified RGCs incubated for 24h in an oxygen/glucose deprived (0.5% oxygen) medium demonstrated no statistically significant differences in cell survival compared to control RGCs maintained under either normoxia or hypoxia. Additionally, immunocytochemical analysis showed increased GAP-43 staining in RGCs after 24h of treatment with s-AMPA (100μM). Conclusions (d): These results indicate that purified RGCs in-vitro are not susceptible to AMPA excitotoxicity as previously hypothesized. Activation of AMPAR increased GAP-43 expression, suggesting AMPAR could possibly increase neurite outgrowth. The ability of AMPA receptors to promote neuroprotection of RGCs remains to be confirmed.
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    (2014-03) Mueller, Brett H.; Park, Yong; Ma, Hai-Ying; Yorio, Thomas
    Purpose (a): Sigma-1 receptor activation and mitogen-activated protein kinases (MAPKs) have been shown to have neuroprotective roles in protecting retinal ganglion cells (RGCs) from cell death. The purpose of this study was to determine if sigma-1 receptor stimulation with pentazocine could promote neuroprotection under conditions of ischemia through the phosphorylation of extracellular signal regulated kinase (pERK)1/2. Methods (b): Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy 1.1 antibody. RGCs were cultured for 7 days before subjecting the cells to an ischemic insult (0.5% oxygen in glucose-free medium) for 6 hours. During the ischemic insult, RGCs were treated with pentazocine (sigma-1 receptor agonist) with or without BD1047 (sigma-1 receptor antagonist). In other experiments primary RGCs were treated with pentazocine, in the presence or absence of PD98059 (ERK1/2 inhibitor). Cell survival/death was assessed by staining with the calcein-AM/ethidium homodimer reagent. Levels of pERK1/2, total ERK1/2, and beta tubulin expression were determined with immunoblotting and immunofluorescence. Results (c): RGCs subjected to an ischemic insult demonstrated more than a 40% increase in cell death, compared to untreated controls. RGCs maintained under ischemia also showed a 50% decrease in expression of pERK1/2 (p<0.05). Cell death was attenuated when RGCs were treated with pentazocine under ischemic conditions and levels of pERK1/2 were increased more than 60% (p<0.05), compared to untreated RGCs subjected to ischemia. Treatment with BD1047 abrogated the pentazocine neuroprotection effects, and also attenuated the increase in levels of pERK1/2 (p<0.05). Finally, treatment with PD98059 also reversed the pentazocine mediated neuroprotective effects on RGCs, and abolished the expression of pERK1/2 (p<0.05). Conclusions (d): These results establish a direct relationship between sigma-1 receptor stimulation and neuroprotective effects under ischemia through the involvement of the MAPK/ERK1/2 pathway in purified RGCs. These findings support a role for sigma receptor agonists as potential neuroprotective agents.
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    (2014-03) Mody, Avani A.; Wordinger, Robert J.; Clark, Abbot F.
    Insight into the BMP4 pathways in various disease models and different cell types has shown BMP4 to be a potent inducer of inhibitor of DNA binding proteins (ID1 and ID3). ID1 and ID3 are negative regulators of basic Helix loop Helix (bHLH) transcription factors and are known to control specific gene expression, including extracellular cellular matrix (ECM) genes. We previously have shown that BMP4 attenuates the pathogenic effects of TGFβ2 in the TM, an ocular tissue involved in regulation of intraocular pressure in glaucoma. We hypothesize that BMP-4 attenuates the effects TGFβ2 in the TM by inducing ID1 and ID3 expression. In our current study, we show that BMP4 induces ID1 and ID3 expression in TM cells. Over-expression and knockdown of ID1 and ID3 in TM cells show that ID1 and ID3 play a crucial role in attenuating the profibrotic effects of TGFβ2 in TM cells. Purpose (a): Increased aqueous humor (AH) outflow resistance causes high intraocular pressure (IOP), which is a critical risk factor in primary open-angle glaucoma. Elevated transforming growth factor b2 (TGFβ2) in the AH of glaucoma patients increases extracellular matrix (ECM) protein deposition in the trabecular meshwork (TM), thereby elevating IOP. Bone morphogenetic protein 4 (BMP4) inhibits the pathogenic effects of TGFβ2 in the TM. However, the underlying molecular mechanism for this BMP4 inhibition remains unknown. BMP4 regulates various cellular processes by induction of inhibitors of DNA binding proteins (ID1, ID3), which are transcriptional regulators that bind specific transcription factors and suppress their functions. This study will determine whether ID1/ID3 are downstream targets of BMP4, attenuating the TGFb-2 effects on TM cells. Methods (b): Cultured primary human TM cells and the GTM3 cell line were treated with BMP4 (5-10ng/ml) for 1-48 hrs. Q-PCR and western immunoblotting were performed to determine ID1 and ID3 expression. GTM3 and primary TM cells were transfected with ID1 and ID3 expression plasmids vectors or ID1 and ID3 siRNA to determine the effects of ID1 and ID3 on TGFβ2 induced extracellular matrix (ECM) proteins. The expression of fibronectin and plasminogen activator inhibitor-1 (PAI-1) was studied by western immunoblotting. Results (c): BMP4 induced ID1 and ID3 expression in TM cells. ID1 and ID3 suppressed the TGFβ2 induction of ECM proteins in TM cells, and therefore are key signaling molecules involved in the BMP4 suppression of TGFβ2 profibrotic activity. These specific regulators controlling TGFβ2 effects in the TM may lead to the development of potential new IOP lowering therapies for the treatment of glaucoma. Conclusions (d): BMP4 induced ID1 and ID3 expression in TM cells. ID1 and ID3 suppressed the TGFβ2 induction of ECM proteins in TM cells, and therefore are key signaling molecules involved in the BMP4 suppression of TGFβ2 profibrotic activity. These specific regulators controlling TGFβ2 effects in the TM may lead to the development of potential new IOP lowering therapies for the treatment of glaucoma.
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    (2014-03) Liu, Xiaobin; Jann, Jamieson; Wu, Hongli
    Oxidative stress is believed to contribute to the pathogenesis of many diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial (RPE) cells are considered as major targets. It is widely accepted that the RPE cells have enormous number of thiol-containing proteins, which can undergo modifications to change retinal protein functions. In contrast, the mechanism of thiol redox regulation in the retina and its association with AMD are still very poorly understood. In particular, the function of glutaredoxin 1 (Grx1), a thiol repair enzyme in cytosol, is virtually unknown. This project seeks to address this paucity in a comprehensive and physiological relevant fashion, and therefore is both novel and innovative. Furthermore, the ability to identify novel therapeutic targets for further research is the first critical step in finding new treatments for AMD. The overall success of this project will raise new hope that Grx1 or its mimic may be a potential therapeutic agent for AMD, and perhaps for other ocular diseases induced by oxidative stress. Purpose (a): The retina is constantly exposed to oxidative stress, which is countered by well-designed antioxidant systems present in retinal pigment epithelial (RPE) cells. Disruption of these systems may lead to the development of age-related macular degeneration (AMD). In this study, we explored the strategy of overexpressing glutaredoxin 1 (Grx1), a component of the endogenous antioxidant defense system, to combat oxidative damage in RPE cells. Methods (b): Human retinal pigment epithelial (ARPE-19) cells were transfected with either a Grx1-containing plasmid or an empty vector. Normal ARPE-19 cells and transfected cells were treated with or without 200 µM H2O2 for 24 h. Grx1 protein expression was detected by western blots and enzyme activity was measured by spectrophotometry. Cell viability was measured by a colorimetric assay with WST8. The morphology of nuclear chromatin was assessed by staining with Hoechst 33342. Apoptosis was quantitatively analyzed by flow cytometry. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Results (c): Grx1 protein level and enzyme activity in Grx1 transfected cells were significantly increased as compared to non-transfected and vector transfected cells. Grx1 overexpression protected ARPE-19 cells from H2O2-induced cell viability loss. Assessment of apoptosis indicated that cells transfected with Grx1 were relatively more resistant to H2O2 with fewer cells undergoing apoptosis as compared to vector control or non-transfected cells. Furthermore, PSSG accumulation was also dramatically attenuated by Grx1 overexpression. Conclusions (d): Grx1 can protect human retinal pigment epithelial cells against H2O2-induced cell death. The mechanism of this protection is likely associated with its ability to prevent lethal accumulation of PSSG.
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    (2014-03) Tovar, Tara; Naik, Monal; Clark, Abbot F.; Wordinger, Robert J.
    Transforming growth factor-beta 2 (TGF-β2) has been implicated in the development of elevated intraocular pressure (IOP) in primary open-angle glaucoma (POAG). Glaucoma patients have increased levels of TGF-β2 in their aqueous humor and trabecular meshwork (TM), and TGF-β2 increases extracellular matrix (ECM) proteins. Thus, we are interested in growth factors that are associated with glaucoma and the ECM. Purpose (a): Transforming growth beta-2 (TGF-β2) has been associated with increased extracellular matrix (ECM) deposition, which is attributed to increased aqueous humor outflow resistance through the trabecular meshwork (TM). We have previously demonstrated that bone morphogenetic protein 1 (BMP1) (an enzyme responsible for the cleavage and maturation of ECM proteins) is expressed and regulated by TGF-β2 in the human TM and that BMP1 regulates lysyl oxidase activity. Also, other factors associated with the ECM remodeling include periostin (POSTN) and procollagen c-endopeptidase enhancer 1 (PCOLCE1). The purpose of this study was to determine whether human TM cells (a) express POSTN and PCOLCE1 (b) whether expression of POSTN and PCOLCE1 are regulated by TGF-β2. Methods (b): Primary human normal (NTM) and glaucomatous (GTM) cells were isolated and subjected to qPCR and Western immunoblotting (WB) for POSTN and PCOLCE1 expression. qPCR was used to determine POSTN and PCOLCE1 expression between control and TGF-β2 treated (5ng/ml for 24 hours) TM cells. WBs of cell lysates and conditioned medium were used to compare POSTN and PCOLCE1 protein expression between control and TGF-β2 treated NTM and GTM cells. Results (c): Human TM cells expressed POSTN and PCOLCE1 mRNA and protein. Exogenous TGF-β2 increased POSTN mRNA expression (p<0.05) and decreased PCOLCE1 expression (p<0.0005) compared to control cells. WB analysis showed increased POSTN secretion in NTM compared to GTM cells (p<0.05). TGF-β2 induced POSTN in NTM cells (p<0.05). However, no POSTN was detected in cell lysates of TM cells. WB analysis showed decreased PCOLCE1 secretion in NTM cells compared to GTM cells (p<0.05). Conclusions (d): POSTN and PCOLCE1 are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of POSTN and PCOLCE1 may lead to structural and functional changes in the ECM within the TM.
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    (2014-03) Nashine, Sonali; Kim, Byung-Jin; Clark, Abbot F.; Pang, Iok-Hou
    Glaucoma is one of the leading causes of blindness and visual impairment, affecting 70 million people worldwide. A major characteristic of this disease is the irreversible death of retinal ganglion cells (RGCs), retinal neurons that transmit visual information from the eye to the brain. There is an unmet need to develop novel therapeutic strategies for glaucoma. One of the major causes of glaucoma is increased pressure inside the eye i.e., increased intraocular pressure. Increased IOP leads to obstruction of the central retinal artery. Obstruction of this artery leads to insufficient blood supply to the eye, which in turn prevents adequate supply of oxygen and nutrients to the eye, causing ischemia. All these events result in improper folding of proteins in the endoplasmic reticulum (ER). Unfolded or misfolded proteins in the ER lead to ER stress which is one of the major pathways of RGC death. C/EBP Homologous protein (CHOP) is a player in this pathway of cell death. We are studying the mechanisms of RGC death in mice. Our mouse model is called the ischemia/reperfusion (I/R) model. In this model, IOP is increased above the normal level for an hour. This prevents blood supply to the eye and leads to ischemia. After an hour, IOP is brought back to normal and blood supply is restored i.e., reperfusion, which causes considerable damage to the eye. Damage caused to the eye in this model simulates the mechanism of RGC death caused in glaucoma. The goal of our project is to investigate if the presence of CHOP in mice causes RGC death after ischemia/reperfusion injury. If it does, then we would study if the absence of CHOP in mice helps in the survival of RGCs. If absence of CHOP in mice is found to protect retinal ganglion cells, then results of the proposed study could lead to the development of a novel therapy for glaucoma. Purpose (a): Retinal ischemia/reperfusion (I/R) causes apoptotic death of retinal ganglion cells (RGC). CHOP is a pro-apoptotic protein and a unfolded protein response (UPR) marker that plays a role in ER-stress mediated apoptotic cell death. The purpose of this study was to investigate the role of CHOP in mouse RGC survival following retinal I/R injury. Methods (b): Retinal I/R was induced in adult C57BL/6J (WT) and CHOP-/- mice by cannulation of the anterior chamber of the left eye with a needle connected to a reservoir of saline. Intraocular pressure was increased to 120 mmHg for 60 min, after which the needle was withdrawn to restore retinal circulation. Uninjured right eyes served as controls. Expression of CHOP protein and other UPR markers (p-eIF2α and BiP) in WT mice post-I/R was studied using Western blot and immunohistochemistry. To compare RGC survival between WT and CHOP-/- mice, retinal flat mount staining with RGC marker, Brn3a was performed. Scotopic threshold response electroretinography (STR-ERG) was performed at 0.03 mcd.s/m2 light intensity to evaluate retinal function. Results (c): CHOP protein was up-regulated by 30 % in I/R injured eyes (1.30 ± 0.11 arbitrary units (a.u.)) compared to control eyes (1 ± 0.07 a.u.) in WT mice three days after I/R injury (p < 0.05). Protein levels of p-eIF2α and BiP also increased by 19% (I/R: 1.19 ± 0.15 a.u., Control: 1 ± 0.06 a.u.) and 11% (I/R: 1.11 ± 0.02 a.u., Control: 1 ± 0.03 a.u.) respectively (both p < 0.05). Co-localization of CHOP and Brn3a confirmed the up-regulation of CHOP specifically in the RGCs. In the uninjured control eyes, CHOP knockout did not affect baseline RGC density or STR-ERG amplitude. I/R injury decreased RGC densities and STR-ERG amplitudes in both WT and CHOP-/- mice. However, survival of RGCs in I/R-injured CHOP-/- mouse eyes (3337.1 ± 316.4 RGC/mm2) was 48% higher (p < 0.05) than that of I/R-injured WT mouse eyes (2248.7 ± 225.9 RGC/mm2) three days after I/R injury. STR-ERG amplitudes were 83 % higher in CHOP-/- I/R eyes (18.6 ± 1.1 μV) compared to WT I/R eyes (10.1 ± 0.9 μV) (p < 0.05). Conclusions (d): Absence of CHOP partially protects against the loss of RGCs and reduction in retinal function (STR-ERG) after I/R injury. These results indicate that CHOP and thus ER-stress play an important role in RGC apoptosis in retinal I/R injury.
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    (2014-03) Choudhury, Shreyasi; Liu, Yang; Clark, Abbot F.; Pang, Iok-Hou
    Glaucoma, a leading cause of blindness worldwide is characterized by injury to the nerve of the eye leading to the death of certain eye cells called retinal ganglion cells (RGCs) and vision loss. Currently available glaucoma therapies only attempt to reduce the eye pressure without addressing the associated RGC death problem. As a result, they do not always sufficiently slow the disease progression in all glaucoma patients. Thus, there is an urgent need to develop strategies for preventing glaucoma associated RGC death. Our preliminary studies have identified a novel protein, caspase-7 as a major player in RGC death pathways. We are studying the role of caspase-7 in RGC death in a mouse glaucoma model and whether it can be targeted for better therapeutic outcome. This project is significant because it will identify a new and potentially critical component of RGC death. This will aid in the design of better therapeutic treatment for glaucoma and other degenerative diseases. Purpose (a): Optic nerve (ON) injury is involved in various ocular diseases, such as glaucoma, which leads to apoptotic death of retinal ganglion cells (RGC) and loss of vision. Caspases have been implicated previously in glaucoma and RGC death. However, the role of caspase-7, a functionally unique caspase, in ON injury and glaucomatous damage has not been studied. Therefore, the purpose of this study is to evaluate the role of caspase-7 in ON injury-induced RGC apoptosis. Methods (b): C57BL/6 (Wt) and caspase-7knockout (casp7KO) mice were used for this study. Optic nerve crush (ONC) was performed on left eyes; right eyes served as control. Western blots of the isolated retinas of Wt mice were used to assess the activation of caspase-7 at 3h, 6h, 12h, 1d, 3d, and 7d after ONC. Immunohistochemistry was performed to detect the localization of caspase-7 in RGC. RGC survival was determined by counting the RBPMS (RGC marker) labeled cells in flat-mounted retinas of Wt and casp7KO mice at 7d, 14d and 28d post injury. Both Wt and casp7KO mice were subjected to spectral-domain optical coherence tomography (SD-OCT) and scotopic threshold response of electroretinography (STR-ERG) to evaluate the retinal structural and RGC functional changes at 7d, 14d, and 28d after ONC. Results (c): Western blot data demonstrated that caspase-7 was activated in Wt retina at 12h, 1d, 3d, and 7d after ONC compared to the uninjured control retinas. The number of surviving RGCs was significantly more (3173±59 cells/mm2, mean±SEM, n=6, p<0.001) in casp7KO retinas compared to Wt retinas (1693±84 cells/mm2) at 28d post ONC. SD-OCT analysis revealed that the thickness of the inner retinal layer (ganglion cell layer, nerve fiber layer, and inner plexiform layer) in casp7KO mice was greater (54±1.1 μm, p<0.05) compared to Wt mice (42.3±1.5 μm). Most importantly, analysis of the STR-ERG response demonstrated a decline in amplitude in Wt ONC eyes (10.5±1.9 μv), whereas the response was significantly higher (20.7±2.3 μv, p<0.05) in casp7KO mice even at 28d post injury. Conclusions (d): The current study indicates that injury to the ON activates caspase-7 and knockout of caspase-7 protects inner retinal layer morphology and RGC function after ONC. Thus, caspase-7 appears to play a critical role in ONC-induced RGC death and inhibition of caspase-7 activity may be a novel therapeutic target for glaucoma and other neurodegenerative diseases of the retina.
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    (2014-03) Olarte, Neal; Silverman, Sean; Wordinger, Robert J.; Clark, Abbot F.
    Glaucoma is an injury to cells of the eye indicated by pressure within the eye which can lead to eventual blindness. Microglial cells are a special type of cell within the central nervous system (CNS) that cleans up cellular damage. Astrocytes are another specialized cell which plays a supportive role important for CNS function. C1q is a part of the immune system usually reserved for clearance of bacteria from the body, but has been recently demonstrated to serve other roles. Microglia and C1q have been implicated in mediating retinal damage in mouse models mimicking glaucoma. This study was conducted to investigate if C1q and either microglia or astrocytes, or both, could be involved in brain damage caused by a simulated glaucoma injury. Purpose (a): Glaucoma is a leading cause of blindness worldwide. Recent studies of glaucomatous retinal injury have observed a correlation of upregulated C1q and increased microglial activity. Using the optic nerve crush (ONC) model of glaucoma, we are investigating whether there is an injurious response involving C1q, microglia, and astrocytes within the superior colliculus (SC), the visual center of the mouse brain. Methods (b): Glaucomatous injury was simulated in mice using ONC of the left eye, while leaving the right eye intact. Brain tissue was harvested at 0, 7, 14, and 28 days post-injury, fixed overnight in 4% paraformaldehyde, and paraffin embedded. Following paraffin removal and antigen recovery, immunohistochemistry was performed to label astrocytes (GFAP), microglia (IBA1), and C1q in the SC. Results (c): Beginning 7 days post-injury, there was an increase in astrocytes, microglia, and C1q, with microglia assuming an activated morphology. Astrocytes and C1q remained elevated through 28 days post-injury, with a gradual reduction in microglial density. These results were observed only within the SC contralateral to the injured nerve, the main target of the retinal ganglion cell (RGC) axons from the ONC eye. Increased C1q and astrocyte activity was not observed in the ipsilateral hemisphere; however, there was a slight increase in microglial density. Conclusions (d): Our data support a similar response in the retina and SC of upregulated C1q, resulting from glaucomatous injury. Microglia and astrocytes also appear to be involved in the acute injury phase. Previous studies of retinal glaucomatous injury have shown that early reduction of C1q is protective. Future studies using a C1q-deficient mouse model might also show protective function against SC glaucomatous injury.